Nature Methods
- 4, 81 - 86 (2007)
Published online: 10 December 2006; | doi:10.1038/nmeth986
Major signal increase in fluorescence microscopy through dark-state relaxation
Gerald Donnert, Christian Eggeling & Stefan W Hell
Max Planck Institute for Biophysical Chemistry, Department of NanoBiophotonics, Am Fassberg 11, 37077 Göttingen, Germany.
Correspondence should be addressed to Stefan W Hell shell@gwdg.de
We report a substantial signal gain in fluorescence microscopy by ensuring that transient molecular dark states with lifetimes >1 s, such as the triplet state relax between two molecular absorption events. For GFP and Rhodamine dye Atto532, we observed a 5–25-fold increase in total fluorescence yield before molecular bleaching when strong continuous-wave or high-repetition-rate pulsed illumination was replaced with pulses featuring temporal pulse separation >1 s. The signal gain was observed both for one- and two-photon excitation. Obeying dark or triplet state relaxation in the illumination process signifies a major step toward imaging with low photobleaching and strong fluorescence fluxes.
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s, such as the triplet state relax between two molecular absorption events. For GFP and Rhodamine dye Atto532, we observed a 5–25-fold increase in total fluorescence yield before molecular bleaching when strong continuous-wave or high-repetition-rate pulsed illumination was replaced with pulses featuring temporal pulse separation >1 
